Molecular and General Genetics MGG

, Volume 242, Issue 2, pp 121–129 | Cite as

Cloning of the blasticidin S deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae

  • Makoto Kimura
  • Takashi Kamakura
  • Quan Zhou Tao
  • Isao Kaneko
  • Isamu Yamaguchi
Original Articles

Abstract

Aspergillus terreus produces a unique enzyme, blasticidin S deaminase, which catalyzes the deamination of blasticidin S (BS), and in consequence confers high resistance to the antibiotic. A cDNA clone derived from the structural gene for BS deaminase (BSD) was isolated by transforming Escherichia coli with an Aspergillus cDNA expression library and directly selecting for the ability to grow in the presence of the antibiotic. The complete nucleotide sequene of BSD was determined and proved to contain an open reading frame of 393 bp, encoding a polypeptide of 130 amino acids. Comparison of its nulceotide sequence with that of bsr, the BS deaminase gene isolated from Bacillus cereus, indicated no homology and a large difference in codon usage. The activity of BSD expressed in E. coli was easily quantified by an assay based on spectrophotometric recording. The BSD gene was placed in a shuttle vector for Schizosaccharomyces pombe, downstream of the SV40 early region promoter, and this allowed direct selection with BS at high frequency, following transformation into the yeast. The BSD gene was also employed as a selectable marker for Pyricularia oryzae, which could not be transformed to BS resistance by bsr. These results promise that the BSD gene will be useful as a new dominant selectable marker for eukaryotes.

Key words

Aspergillus terreus Blasticidin S deaminase gene Selectable marker Schizosaccharomyces pombe Pyricularia oryzae 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Amann E, Ochs B, Abel K-J (1988) Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69:301–315Google Scholar
  2. Ballance DJ (1986) Sequences important for gene expression in filamentous fungi. Yeast 2:229–236Google Scholar
  3. Endo T, Furuta K, Kaneko A, Katuki T, Kobayashi K, Azuma A, Watanabe A, Shimazu A (1987) Inactivation of blasticidin S by Bacillus cereus I. inactivation mechanisms. J Antibiot (Tokyo) 40:1791–1793Google Scholar
  4. Endo T, Kobayashi K, Nakayama N, Tanaka T, Kamakura T, Yamaguchi I (1988) Inactivation of blasticidin S by Bacillus cereus II. isolation and characterization of a plasmid, pBSR8, from Bacillus cereus. J Antibiot (Tokyo) 41:271–273Google Scholar
  5. Gmünder H, Kohli J (1989) Cauliflower mosaic virus promoters direct efficient expression of a bacterial G418 resistance gene in Schizosaccharomyces prombe. Mol Gen Genet. 220:95–101Google Scholar
  6. Huang KT, Misato T, Asuyama H (1964) Effect of blasticidin S on protein synthesis of Pyricularia oryzae. J Antibiot Ser A 17:65–70Google Scholar
  7. Itaya M, Yamaguchi I, Kobayashi K, Endo T, Tanaka T (1990) The blasticidin S resistance gene (bsr) selectable in a single copy state in the Bacillus subtilis chromosome. J Biochem 107:799–801Google Scholar
  8. Izumi M, Miyazawa H, Kamakura T, Yamaguchi I, Endo T, Hanaoko F (1991) Blasticidin S resistance gene (bsr): a novel selectable marker for mammalian cells. Exp Cell Res 197:229–233Google Scholar
  9. Kamakura T, Kobayashi K, Tanaka T, Yamaguchi I, Endo T (1987) Cloning and expression of a new structural gene for blasticidin S deaminase, a nucleoside aminohydrolase. Agric Biol Chem 51:3165–3168Google Scholar
  10. Kamakura T, Yoneyama K, Yamaguchi I (1990) Expression of the blasticidin S deaminase gene (bsr) in tabacco: fungicide tolerance and a new selective marker for transgenic plants. Mol Gen Genet 223:332–334Google Scholar
  11. Kistler HC, Benny UK (1988) Genetic transformation of the fungal pathogen, Fusarium oxysporum. Curr Genet 13:145–149Google Scholar
  12. Kobayashi K, Kamakura T, Tanaka T, Yamaguchi I, Endo T (1991) Nucleotide sequence of the bsr gene and N-terminal amino acid sequence of blasticidin S deaminase from blasticidin S resistant Escherichia coli TK121. Agric Biol Chem 55:3155–3157Google Scholar
  13. Kudla B, Persuy MA, Gaillardin C, Heslot H (1988) Construction of an expression vector for the fission yeast Schizosaccharomyces pombe. Nucleic Acids Res 16:8603–8617Google Scholar
  14. Lang-Hinrichs C, Dossereck C, Fath I, Stahl U (1990) Use of Tn903 neomycin-resistance gene for promoter analysis in the fission yeast Schizosaccharomyces pombe. Curr Genet 18:511–516Google Scholar
  15. Moreno S, Klar A, Nurse P (1991) Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. In: Guthrie C, Fink GR (ed) Methods Enzymol 194:795–823Google Scholar
  16. Mullaney EJ, Hamer JE, Roberti KA, Yelton MM, Timberlake WE (1985) Primary structure of the trpC gene from Aspergillus nidulans. Mol Gen Genet 199:37–45Google Scholar
  17. Okayama H, Berg P (1983) A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol Cell Biol 3:280–289Google Scholar
  18. Okazaki K, Okazaki N, Kume K, Jinno S, Tanaka K, Okayama H (1990) High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by transcomplementation of Schizosaccharomyces pombe. Nucleic Acids Res 18:6485–6489Google Scholar
  19. Otake N, Takeuchi S, Endo T, Yonehara H (1966) Chemical studies on blasticidin S. III. The structure of blasticidin S. Agric Biol Chem 30:132–141Google Scholar
  20. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  21. Scharf SJ (1990) Cloning with PCR. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols. Academic Press, San Diego pp 84–91Google Scholar
  22. Song BH, Neuhard J (1989) Chromosomal location, cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase. Mol Gen Genet 216:462–468Google Scholar
  23. Staben C, Jensen B, Singer M, Pollock J, Schechtman M, Kinsey J, Selker E (1989) Use of a bacterial hygromycin B resistance gene as a dominant selectable marker in Neurospora crassa transformation. Fungal Genet Newslett 36:79–81Google Scholar
  24. Takeuchi S, Hirayama K, Ueda K, Yonehara H (1958) Blasticidin S, a new antibiotic. J Antibiot Ser. A 11:1–5Google Scholar
  25. Yamaguchi H, Tanaka N (1966) Inhibition of protein synthesis by blasticidin S. II. Studies on the site of action in E. coli polypeptide synthesizing systems. J Biochem 60:632–634Google Scholar
  26. Yamaguchi I, Misato T (1985) Active center and mode of reaction of blasticidin S deaminase. Agric Biol Chem 49:3355–3361Google Scholar
  27. Yamaguchi I, Takagi K, Misato T (1972) The fate of blasticidin S, an agricultural antibiotic, in the environment. I. The site for degradation of blasticidin S. Agric Biol Chem 36:1719–1727Google Scholar
  28. Yamaguchi I, Shibata H, Seto H, Misato T (1975) Isolation and purification of blasticidin S deaminase from Aspergillus terreus. J Antibiot (Tokyo) 28:7–14Google Scholar
  29. Yamaguchi I, Seto H, Misato T (1986) Substrate binding by blasticidin S deaminase, an aminohydrolase for novel 4-aminopyrimidine nucleosides. Pestic Biochem Physiol 25:54–62Google Scholar
  30. Yanai K, Horiuchi H, Takagi M, Yano K (1991) Transformation of Rhizopus niveus using a bacterial blasticidin S resistance gene as a dominant selectable marker. Curr Genet 19:221–226Google Scholar
  31. Yelton MM, Hamer JE, Timberlake WE (1984) Transformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci USA 81:1470–1474Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Makoto Kimura
    • 1
  • Takashi Kamakura
    • 2
  • Quan Zhou Tao
    • 1
  • Isao Kaneko
    • 1
  • Isamu Yamaguchi
    • 1
  1. 1.Microbial Toxicology LaboratoryInstitute of Physcial and Chemical Research (RIKEN)Wako-shi, SaitamaJapan
  2. 2.Biodesign Research GroupInstitute of Physical and Chemical Research (RIKEN)Wako-shi, SaitamaJapan

Personalised recommendations